Method of screening endothelial cells for angiogenic capability

ABSTRACT

Provided is a method of screening a primary endothelial cell population for angiogenesis capability comprising: (a) measuring the percentage of cells that are positive for VEGF R2 and CD34, the level of VEGF R2, or measuring the VEGF R2 to VEGF R1 ratio in the population; and (b) selecting those populations where the measured percentage or the measured ratio is over a threshold value.

This application is a divisional application of U.S. patent applicationSer. No. 10/913,209, filed Aug. 6, 2004, now U.S. Pat. No. 7,547,518,which claims priority of U.S. Provisional Patent Application No.60/496,308, filed Aug. 19, 2003, which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally directed to methods of identifyinghighly angiogenic endothelial cells. In particular, the presentinvention is directed to a method of screening endothelial cells forangiogenic capability comprising quantifying the endothelial cells thatare VEGF R2 positive and CD34 positive, the VEGRF 2 amount, ordetermining the VEGF R2 to VEGF R1 ratio or number of VEGF-R2 positivecells to number of VEGF-R1 positive cells.

2. Background of the Invention

Endothelial cells that form the lining of blood vessels are well knownfor their capacity to adjust their numbers and arrangement to suitelocal requirements. All tissues depend on a blood supply and the bloodsupply depends on endothelial cells. Blood vessels create an adaptablelife support system in every region of the body. If not for endothelialcells extending and maintaining this network of blood vessels, tissuegrowth and repair would not be possible.

The largest blood vessels are arteries and veins, which have a thicktough outer wall of connective tissue and smooth muscle. The wall islined by a thin single layer of endothelial cells, separated from thesurrounding outer layers by a basal lamina. While the amounts ofconnective-tissue and smooth muscle in the vessel, wall may varyaccording to the vessel's diameter and function, the endothelial liningis always present. In the smaller capillaries and sinusoids, the wallsconsist solely of endothelial cells and basal lamina. Thus, endothelialcells line the entire vascular system. Studies have shown that arteriesand veins develop from small vessels constructed solely of endothelialcells and a basal lamina, connective tissue and smooth muscle beingadded later where required upon signals from the endothelial cells.

Throughout the vascular system endothelial cells retain a capacity forcell division and movement. The is important in repair and maintenanceof the vascular system. For example, if a part of the wall of a bloodvessel is damaged and loses endothelial cells, neighboring endothelialcells will proliferate and migrate in to cover the exposed surface.Newly formed endothelial cells have also been know to cover the innersurface of plastic tubing used by surgeons to replace damaged bloodvessels.

Endothelial cells not only repair damaged blood vessels, they alsocreate new blood vessels. The do this in embryonic tissues to supportgrowth, in normal adult tissue for repair and maintenance, and indamaged tissue to support repair. This process is called angiogenesis.

Angiogenesis is the fundamental process by which new blood vessels areformed. This process is essential in numerous normal physiologicalphenomena such as embryonic development, tissue growth, tissueremodeling, and wound healing. Angiogenesis is also important in certainpathological events. In addition to a role in solid tumor growth andmetastasis, other notable conditions with an angiogenic component arearthritis, psoriasis and diabetic retinopathy. In other pathologies suchas the cardiovascular diseases, the diseases of the peripheral arteriesas well as the vascular and cerebral lesions, angiogenesis can presentan important therapeutic base. The promotion of angiogenesis in thedamaged locations can lead to formation of sanguineous neovessels thatare lateral and alternative to the damaged vessels, thereby providingblood and, thus, oxygen and other nutritive and biological factorsnecessary for the survival of the tissues involved.

During the process of angiogenesis, endothelial cells, which exist in aquiescent state as part of an existing blood vessel, grow and enter amigratory, proliferative state. This migratory, proliferative state iseventually resolved when the cells differentiate into capillary tubesand return to the quiescent state as part of a functional new bloodvessel. The process of angiogenesis is orchestrated by a complex networkof multiple macromolecular interactions. Some essential angiogenicfactors include fibroblast growth factor-basic (bFGF), vascularendothelial growth factor (VEGF), the angiopoietins, cytokines,extracellular matrix proteins, and matrix metalloproteases. Thesefactors are produced locally by stromal cells (e.g., smooth musclecells, pericytes, fibroblasts) and by activated leukocytes that arerecruited to the area. A feature of endothelial cells is their capacityto undergo tubulogenesis, an aspect of angiogenesis, under appropriateconditions.

Angiogenesis is regulated in both normal and malignant tissues by thebalance of angiogenic stimuli and angiogenic inhibitors that areproduced in the target tissue and at distant sites. Vascular endothelialgrowth factor-A (VEGF, also known as vascular permeability factor, VPF)is a primary stimulant of angiogenesis. VEGF is a multifunctionalcytokine that is induced by hypoxia and oncogenic mutations and can beproduced by a wide variety of tissues.

Angiogenesis is stimulated and harnessed by some neoplasms (e.g.,tumors) to increase nutrient uptake. However, in contrast to normalangiogenesis, which leads to anastomoses (i.e., vessel connections) andcapillary maturation, angiogenesis associated with neoplasia is acontinuous process. Endothelial cells are activated by nearby neoplasticcells to secrete not only VEGF which stimulates angiogenesis, but alsomatrix metalloproteases (MMP) which degrade the surroundingextracellular matrix. The endothelial cells then invade theextracellular matrix where they proliferate, migrate, and organize toform new blood vessels, which support neoplasm growth and survival.

The newly vascularized neoplasm continues to grow, leading to furthernutrient deprivation and chronic pro-angiogenic signaling. Thevasculature of neoplasms is characterized by the presence of structuralirregularities (lacunae) and a low rate of formation of inter-vesselconnections. This partially dysfunctional vasculature fuels thepermanent requirement for angiogenesis. Additionally, this incompletevasculature is believed to promote the shedding of neoplastic cells intothe systemic circulation. Hence, the angiogenic potential of a neoplasmcorrelates with metastatic potential. As a significant proportion ofneoplasms are dependent on continued angiogenesis, inhibition ofangiogenesis blocks neoplasm growth which often leads to completenecrosis of the neoplasm.

The interplay of growth factors (e.g., VEGF) and surface proteinextracellular interactions drive the process of angiogenesis through apredictable sequence of events. Activation of endothelial cells bypro-angiogenic stimuli results in vasodilation, hyperpermeability, andlocal release of proteases which degrade the basement membrane andextracellular membrane (ECM). This allows the formation of a provisionalfibrin matrix, which provides a primary scaffold for the assembly ofearly microvessels. Motogenic endothelial cells sprout into the matrixand migrate with controlled matrix degradation at the tip. Proliferationoccurs proximal to migration with formation of a primitive tube.Extensive remodeling ensues until the new capillary matures andanastomoses (i.e., fuses and joins) with other sprouts.

VEGF is critical to the angiogenesis process. VEGF induces angiogenesisand endothelial cell proliferation and it plays an important role inregulating vasculogenesis. VEGF is a heparin-binding glycoprotein thatis secreted as a homodimer of 45 kDa. Most types of cells, but usuallynot endothelial cells themselves, secrete VEGF. VEGF is known toincrease vascular permeability and cause vasodilatation, partly throughstimulation of nitric oxide synthase in endothelial cells. VEGF can alsostimulate cell migration and inhibit apoptosis. There are threereceptors in the VEGF receptor family (VEGF R1, VEGF R2 and VEGF R3).These receptors have the common properties of multiple immunoglobulin G(IgG)-like extracellular domains and tyrosine kinase activity.Endothelial cells also express additional VEGF receptors, Neuropilin-1and Neuropilin-2. VEGF-A is the most commonly occurring VEGF. VEGF-Bthrough VEGF-D are lesser occurring. VEGF-A binds to VEGF R1 and VEGF R2and to Neuropilin-1 and Neuropilin-2. Placental Growth Factor (PIGF) andVEGF-B bind VEGF R1 and Neuropilin-1. VEGF-C and -D bind VEGF R3 andVEGF R2. VEGF R1 and VEGF R2 are upregulated on tumor and proliferatingendothelium, partly by hypoxia and also in response to VEGF-A itself.VEGF R1 and VEGF R2 can interact with multiple downstream signalingpathways via proteins such as PLC; Ras, Sic, Nck, PKC and PI3-kinase.VEGF R1 is of higher affinity than VEGF R2 and mediates motility andvascular permeability. VEGF R2 is necessary for proliferation.

The therapeutic implications of angiogenic growth factors were firstdescribed by more than 30 years ago. (Folkman, N. Engl. J. Med.,85:1182-1186 (1971)). Recent work has established the feasibility ofusing recombinant angiogenic growth factors, such as vascularendothelial growth factor (VEGF-1) to expedite and/or augment collateralartery development in animal models of myocardial and hindlimb ischemia(Takeshita, et al., Circulation, 90:228-234 (1994) and Takeshita, etal., J Clin Invest, 93:662-70 (1-994)). However, alternative methods forpromoting angiogenesis are desirable for a number of reasons. Forexample, it is believed that native endothelial cell number and/orviability decreases over time. Thus, in certain patient populations,e.g., the elderly, cells capable of responding to angiogenic proteinsmay be limited. These problems can be reduced by administering isolatedendothelial cells to patients undergoing treatment for ischemic disease.However, prior to the present invention, such methods were prohibitivelyexpensive as they required expensive isolation and maintenance of cells.

Angiogenesis or angiogenic capability refers to the capacity of anendothelial cell to generate new capillary blood vessels. Highlyangiogenic endothelial cells have a wide spectrum of uses including, usein the prevention or treatment of trauma, graft rejection,cerebrovascular ischemia, renal ischemia, pulmonary ischemia, ischemiarelated to infection, limb ischemia, ischemic cardiomyopathy,cerebrovascular ischemia, and myocardial ischemia. Impacted tissue canbe associated with nearly any physiological system including thecirculatory system or the central nervous system, e.g., a limb, graft(e.g., muscle or nerve graft), or organ (e.g., heart, brain, kidney andlung). Highly angiogenic endothelial cells may also be utilized inangiogenesis assay kits and in the study of endothelial cells,particularly the functions and permeability of the endothelial cellbarrier. In order to determine angiogenic capability and the ability ofendothelial cells to form tubes it has been necessary to carry outangiogenesis assays for each lot of endothelial cells. These assays aretime consuming and costly. Therefore, there is a need for a cheaper,quicker way to screen for and mark angiogenesis capability amongendothelial cells. This need is met by the present invention whichprovides an effective and cost-efficient means of identifying highlyangiogenic endothelial cells.

SUMMARY OF THE INVENTION

The present invention is directed to methods of screening endothelialcell populations for angiogenic capability. One particular embodiment ofthe invention is directed to a method of screening a primary endothelialcell population for angiogenesis capability comprising measuring thepercentage of cells that are positive for VEGF R2 and CD34, the level ofVEGF R2, or measuring the VEGF R2/VEGF R1 ratio in the population; andselecting those populations where the measured percentage or measuredratio is over a threshold value. The step of measuring the percentage ofcells that are positive for VEGF R2 and CD34 or measuring the VEGFR2/VEGF R1 ratio in the population can, in a preferred embodiment,comprise staining the endothelial cell population with anti-VEGF R2antibodies and anti-CD34 antibodies or anti-VEGF R2 and anti-VEGF R1antibodies.

DETAILED DESCRIPTION OF THE INVENTION

For simplicity and illustrative purposes, the principles of the presentinvention are described by referring to various exemplary embodimentsthereof. Although the preferred embodiments of the invention areparticularly disclosed herein, one of ordinary skill in the art willreadily recognize that the same principles are equally applicable to,and can be implicated in other compositions and methods, and that anysuch variation would be within such modifications that do not part fromthe scope of the present invention. Before explaining the disclosedembodiments of the present invention in detail, it is to be understoodthat the invention is not limited in its application to the details ofany particular embodiment shown, since of course the invention iscapable of other embodiments. The terminology used herein is for thepurpose of description and not of limitation. Further, although certainmethods are described with reference to certain steps that are presentedherein in certain order, in many instances, these steps may be performedin any order as may be appreciated by one skilled in the art, and themethods are not limited to the particular arrangement of steps disclosedherein.

Endothelial cells are the cells that make up the inside of bloodvessels. Human and other mammalian endothelial cells are preferred foruse in the invention. These include but are not limited to human dermalmicrovascular endothelial cells and human pulmonary microvascularendothelial cells (HMVEC) and human umbilical vein endothelial cells(HUVEC).

Angiogenesis capability refers to the capacity of an endothelial cell togenerate new capillary blood vessels. An aspect of this capacity is theability of the endothelial cell to form tubes. This tube-forming isreferred to as tubulogenesis.

The present invention is directed, among other things, toward a methodof screening endothelial cells for angiogenesis capability using ananti-VEGF antibody. The term “anti-VEGF antibody” refers to an antibodythat blocks VEGF activity. Examples of such antibodies the antibodies tohuman VEGF described by Kim, U.S. Pat. No. 6,582,959, or those describedby Thorpe et al., U.S. Pat. No. 6,524,583.

In a preferred embodiment the method of screening endothelial cellscomprises measuring the percentage of VEGF R2 positive and CD34 positivecells in each endothelial cell population or measuring the ratio of VEGFR2/VEGF R1 in the population. Populations where the measured percentageor ratio is over a threshold are then selected. These thresholds can bedetermined with ordinary experimentation correlating the amount or ratiowith the desired tubule-forming quality of the cell population.

The VEGF R2 positive cells in each endothelial cell population can bequantified using various known methods, including but not limited tofluorescence activated cell sorting (FACS) analysis, immunofluorescentimaging and data processing or cell-based Enzyme-Linked ImmunosorbentAssay (ELISA). Antibodies for use in such methods are well known andreadily available, e.g., antibodies directed to vascular endothelialgrowth factor receptors can be identified and obtained from a variety ofsources, such as the MSRS catalog of antibodies (Aerie Corporation,Birmingham, Mich.), and can also be prepared via conventional antibodygeneration methods.

It is believed that the more VEGF R2 positive and CD34 positiveendothelial cells, or the higher VEGF R2 to VEGF R1 ratio, the higherthe capability of the endothelial cells to form tubes and the higher theangiogenic capability of the endothelial cells.

In one embodiment of the present invention the method of screeningendothelial cells for angiogenic properties comprises staining the humanendothelial cells with anti-VEGF R2 receptors and anti-CD34 andanti-VEGF R1 receptors. In a preferred embodiment after the step ofstaining the endothelial cells the stained cells are labeled withsecondary antibodies. The secondary antibodies are preferablyfluorescein or rhodamine conjugated. In one embodiment the stained andlabeled endothelial cells are examined using, for example, FACS,fluorescent imaging, or ELISA and the VEGF R2 positive and CD34 positivecells are identified. In a preferred embodiment the percentage of VEGFR2 positive and CD34 positive cells in each endothelial cell populationor ratio of VEGF R2/VEGF R1 in the population is measured. Endothelialpopulations are selected such that the percentage of VEGF R2 positiveand CD34 positive cells in each endothelial cell population or the ratioof VEGF R2/VEGF R1 in the population is over a certain threshold.

The method of selecting cell populations can also be incorporated into amethod of identifying bioactive agents by contacting prospectivebioactive agents with the endothelial cells at some time relevant tomodulating angiogenic activity. Bioactive agents include substances suchas chemicals that can act on a cell, tissue, organ or organism,including but not limited to insecticides or drugs (i.e.,pharmaceuticals) to create a change in the functioning of the cell,organ or organism. The time frame can be readily determinedexperimentally. Bioactive agents that reduce angiogenesis are candidatebioactive agents for disrupting blood cell formation sought to beinduced by tumor cells. Bioactive agents that stimulate angiogenesis arecandidate bioactive agents to increase vascularization in tissue damagedby ischemic events, tissue whose vascularization has been damaged byenvironmental factors such as smoking, or pathological factors resultingfrom, e.g., cardiovascular diseases, diseases of the peripheral arteriesas well as vascular and cerebral lesions. The selection method isbelieved to be particularly applicable to angiogenesis assays thatmeasure tubule formation, though other angiogenesis measures, such ascell migration, can be used. Useful endothelial cell tube formationassays include tube formation on matrices such as Matrigel(Becton-Dickinson, Franklin Lakes, N.J.), fibrin gel or collagen gel andtube formation in co-culture models such endothelial cells co-culturewith either tumor cells or normal fibroblast cells. For example, primaryhuman endothelial cells (such as HUVEC and HMVEC cells) can be mixed(e.g., at a ratio of 100:1) with tumor cell U251 (a glioblastoma cellline). The cells can be seeded in 6-well plates at a density of, forexample, 1×10e6/well. The co-cultures can be incubated in endothelialcell medium EGM-2 MV (Cambrex, Walkersville, Md.) in a 37° C./5% CO2incubator and the medium replaced every two days till day 11. Tubeformation should be observed on and after day 7.

The method of the invention can further be used to adjust the angiogenicpotential of cell populations by mixing populations to achieve thedesired amounts or ratio of the markers used in the invention. Or,separation devices such as fluorescence activated cell sorting devicescan be used to isolate cell populations enriched in cells expressingVEGF R2 receptor and CD34. The enriched populations can, in turn, beused to adjust the amounts or ratio of the cells expressing the markersused in the invention.

While the invention has been described with an emphasis on particularembodiments thereof, those skilled in the art may make variousmodifications to the described embodiments of the invention withoutdeparting from the scope of the invention. Although the invention hasbeen described and disclosed in various terms and certain embodiments,the scope of the invention is not intended to be, nor should it bedeemed to be, limited thereby and such other modifications orembodiments as may be suggested by the teachings herein are particularlyreserved, especially as they fall within the breadth and scope of theclaims here appended. Those skilled in the art will recognize that theseand other variations are possible within the scope of the invention asdefined in the following claims and their equivalents.

1. A method of selecting for higher angiogenesis capability amongendothelial cells in an endothelial cell population comprising: a)staining the endothelial cell population with one or more anti-VEGF R2antibodies and one or more anti-CD34 antibodies; b) measuring percentageof endothelial cells that are positive for VEGF R2 and CD34; and c)selecting those populations having more VEGF R2 positive and CD34positive endothelial cells, wherein the more VEGR R2 positive and CD34positive endothelial cells, the higher the capability of the endothelialcells to form tubes and the higher the angiogenic capability of theendothelial cells.
 2. The method of claim 1, wherein the step ofmeasuring the percentage of endothelial cells that are positive for VEGFR2 and CD34 in the stained population comprises fluorescence activatedcell sorting analysis.
 3. The method of claim 1, wherein the step ofmeasuring the percentage of endothelial cells that are positive for VEGFR2 and CD34 in the stained population comprises immunofluorescentimaging and data processing.
 4. The method of claim 1, wherein the stepof measuring the percentage of endothelial cells that are positive forVEGF R2 and CD34 in the stained population comprises a cell-basedEnzyme-Linked immunosorbent Assay.
 5. The method of claim 1, furthercomprising labeling the endothelial cell population with one or moresecondary antibodies.
 6. The method of claim 5, wherein the one or moresecondary antibodies are fluorescein or rhodamine conjugated.
 7. Themethod of claim 1, wherein the endothelial cell population compriseshuman dermal microvascular endothelial cells.
 8. The method of claim 1,wherein the endothelial cell population comprises human pulmonarymicrovascular endothelial cells.
 9. The method of claim 1, wherein theendothelial cell population comprises human umbilical vein endothelialcells.